Downloaded from the KEGG GENES Database [9]

PH Y L O G E N E T I C A N A L YSIS O F LIPID M E DI A T O R GPC RS SAYAKA MIZUTANI1 MICHIHIRO TANAKA1 [email protected] [email protected] CRAIG E. WHEELOCK2 MINORU KANEHISA1,3 SUSUMU GOTO1 [email protected] [email protected] [email protected] 1 Bioinformatics Center, Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0021, Japan 2 Department of Medical Biochemistry and Biophysics, Division of Physical Chemistry II, Karolinska Institutet, Stockholm, 171-77, Sweden 3 Human Genome Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Lipid mediators is the collective term for prostanoids, leukotrienes, lysophospholipids, platelet- activating factor, endocannabinoids and other bioactive lipids, that are involved in various physiological functions including inflammation, immune regulation and cellular development. They act autocrinically and paracrinically by binding to their ligand-specific G-protein coupled receptors *3&5V 6LQFH¶V a number of lipid GPCRs have been cloned in humans, with a few more identified in other vertebrates. However, the conservation of these receptors has been poorly investigated in other eukaryotes. Herein we performed a phylogenetic analysis by collecting their orthologs in 13 eukaryotes with complete genomes. The analysis shows that orthologs for prostaglandin receptors are likely to be conserved in the 13 eukaryotes. In contrast, those for lysophospholipid and cannabinoid receptors appear to be conserved in only vertebrates and chordates. Receptors for leukotrienes and other bioactive lipids are limited to vertebrates. These results indicate that the lipid mediators and their receptors have coevolved with the development of highly modulated physiological functions such as immune regulation and the formation of the central nervous system. Accordingly, examining the presence and role of lipid mediator GPCR orthologs in invertebrate species can provide insight into the development of fundamental biological processes across diverse taxa. Keywords: lipid mediators, eicosanoids, GPCRs, phylogenetic analysis, eukaryotes, invertebrates 1. Introduction Lipid mediators is a collective term for prostanoids, leukotrienes (LTs), lysophospholipids, platelet-activating factor (PAF), endocannabinoids (CB), and other bioactive lipids. These mediators have a range of biological activities and are important in multiple fundamental biological processes including reproduction, signaling processes, immune function and disease etiology and pathogenesis to name a few. Lipid mediators are synthesized from precursor membrane lipids (e.g., arachidonic acid) and have relatively short half-lives, which limits their signaling functions to be autocrinic and paracrinic. These mediators act on Class I G protein-coupled receptors (GPCRs) [18]. 1 2 Eicosanoids are a major class of lipid mediators that are the oxygenated products of arachidonic acid [17, 5]. Following its release from membrane phospholipids, arachidonic acid can be modified by 3 distinct enzymatic pathways including cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (P450) pathways. Eicosanoids are further divided into subgroups based upon these pathways, with prostaglandins (PGs) being synthesized via COX-dependent pathways in combination with terminal synthases and LTs being synthesized via LOX-dependent pathways. The different eicosanoids bind to distinct GPCRs that demonstrate receptor-specific binding affinities. There are currently 5 known prostanoid receptors: EP FP, DP, IP and TP, which preferentially respond to PGE2, 3*)Į, PGD2 and PGI2 and thromboxane A2 (TXA2), respectively. The EP group is further divided into 4 subtypes termed EP1-4. The physiological effects of each PG have been well-studied in mammals [5]. PGE2 and PGI2 induce several inflammatory responses including fever, pain, and vasodilatation.. 3*)Į and PGE2 have essential roles in reproduction including oocyte maturation and uterine contraction in mammals [22]. TXA2 acts in platelet aggregation. To date, four different LT receptors have been identified: cysteinyl leukotriene receptor 1 and 2 (CysLT1/2) and leukotriene B4 (LTB4) receptor 1 and 2 (BLT1/2). The primary ligands for CysLT1/2 are the glutathione peptide conjugated LTs (LTC4, LTD4 and LTE4) and the main ligand for the BLT1/2 is LTB4. However, recently, a COX- catalyzed product 12-HHT (12S-hydroxy-5Z,8E,10E-heptadecatrienoic acid) was found to be an endogenous ligand of BLT2 [13]. Leukotriene receptors are highly expressed in neutrophils and eosinophils, and are involved in the chemotasis of these cells to the inflamed site. Recently it has been elucidated that they are also expressed in immune cells, regulating immune responses [24]. Lysophopholipids are structurally categorized into lysophosphatidic acids (LPAs) and sphingosine 1-phosphates (S1Ps), both of which generate a large number of species depending on the sn-1 and sn-2 positions of the lipid architecture and the positions and the number of double bonds in the acyl chains. Recently 6 distinct receptors for LPAs, LPA1-6, and five for S1Ps, S1P1-5, have been identified. Lysophopholipids are involved in the regulation of cellular responses including cell proliferation, differentiation, migration, adhesion and morphogenesis. Several pathological studies have revealed their essential roles in the formation of the central nervous system, angiogenesis and the regulation of vascular contraction [2, 11, 19]. S1Ps are also involved in the regulation of lymphocyte migration [14]. Finally, two CB receptors, CB1 and CB2, have been identified; CB1 is expressed mainly in the central nervous system and CB2 in immune cells [18]. Most GPCR-based studies to date have been mostly limited to humans and rodents. Accordingly, the conservation of these receptor genes and their functions are poorly understood in other eukaryotes. To address this point, we performed a phylogenetic analysis to investigate the variation of lipid GPCRs in 13 eukaryotic organisms in an attempt to correlate the emergence of lipid signaling functions to the development of physiological activities in which they are involved. 3 2. Materials and Methods 2.1. Obtaining experimentally characterized lipid mediator GPCRs 46 amino acid sequences (26 in H. sapiens, 10 in M. musculus, seven in R. norvegicus, two in X .laevis and one in D. rerio) of experimentally annotated lipid mediator GPCRs were obtained from the UniProt database [25]. The UniProt IDs and annotations of the query sequences are listed in Table 1. 2.2. BLAST search for lipid mediator GPCR candidates Using the query sequences we performed a BLAST search [1] for 16 eukaryotic organisms with complete genomes, H. sapiens (hsa), M. musculus (mmu), R. norvegicus (rno), X. laevis (xla), D. rerio (dre), C. intestinalis (cin), B. floridae (bfo), S. purpuratus (spu), D. melanogaster (dme), C. elegans (cel), B. malayi (bmy), N. vectensis (nve), T. adhaerens (tad), A. thaliana (ath), S. cerevisiae (sce) and M. brevicollis (mbr). Amino acid sequences of the 16 organisms were downloaded from the KEGG GENES database [9]. Since a large part of the search results consisted of olfactory receptor sequences, each of the 46 search results was given an E-value optimally small enough to remove all known olfactory receptors, based on an assumption that candidate lipid GPCRs have higher degrees of similarity with the queries than olfactory receptors. 2.3. Identifying ortholog candidates using phylogenetic trees For each of the organisms the refined search results were merged with the 46 lipid query sequences. The merged sequences were used to perform a multiple sequence alignment with the program E-INS-I in MAFFT version 5.8 [10] and to construct a rooted phylogenetic tree using the neighbor-joining (NJ) method [16] using the program QuickTree [7] with bootstrap values. Nine human olfactory receptor sequences were used as an outgroup in order to identify the root of the tree. In order to identify ortholog candidates, query sequences were grouped into seven classes, prostanoid receptors (PG), leukotriene receptors (LT), fatty acid receptors (FA), lysophospholipid receptors (LPA & S1P), cannabinoid receptors (CB), platelet-activating factor receptors (PAF), bile acid receptors (BA) and non-lipid GPCRs. In this process subsets that belong to the same class, such as receptors for PGE2 and 3*)Į in the prostanoid receptor class (PG), were not distinguished from each other. Since the BLAST search results contained many non-lipid GPCRs whose similarities are higher than olfactory receptors, we also considered 16 known human non-lipid receptors, which were also in the BLAST hits, so that the annotated part of the search results were increased. On the constructed trees ortholog candidates of the query sequences were manually extracted by the procedures (i) and (ii). (i) For each class of queries, define ortholog cluster(s) via the following two steps; 4 Step 1. For each query, if the nearest query node(s) belongs to the same class, all the internal candidates are considered as orthologs. Step 2. Repeat Step 1 until no such nearest queries are found. (ii) To expand each ortholog cluster defined in (i), candidates in the sibling cluster are tested for validity by BLAST search against the human genome; if queries in the defined ortholog cluster are listed as the top hits, the candidates are considered as orthologs. Fig. 1 shows the overall flow chart of the methods described

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